Delta 4,20,22-bufatrienolide rhamnoside ethers and process for their preparation

ABSTRACT

CARDIOACTIVE $4,20,22-BUFATRIENOLIDE RHAMNOSIDE ETHERS OF THE FORMULA   10-R,3-((3-(R1-O-),4-(R2-O-),5-(R3-O-),6-CH3-TETRAHYDRO-   PYRAN-2-YL)-O-),14-HO,17-(2-(O=)PYRAN-5-YL)-ESTR-4-ENE   WHEREIN R IS METHYL OR FORMYL,R1 AND R2 TAKEN ALONE ARE HYDROGEN, LOWER ALKYL, OR ACETYL, R1 AND R2 TAKEN TOGETHER ARE   &gt;C(-CH3)2   R3 IS HYDROGEN, LOWER ALKYL, OR ACETYL, AND WHEREIN AT LEAST ONE OF R1, R2, OR R3 IS ALKYL, AND THE METHOD OF PREPARING THEM BY REACTION OF THE CORRESPONDING 2&#39;&#39;,3&#39;&#39;,4&#39;&#39;HYDOXY COMPOUNDS OR 2&#39;&#39;,3&#39;&#39;-ISOPROPYLIDENE-4&#39;&#39;-HYDROXY COMPOUNDS WITH AN ALKYL HALIDE, OPTIONALLY FOLLOWED BY HYDROLYSIS OF THE ISOPROPYLIDENE GROUP AND/OR ACETYLATION OF UNETHERIFIED HYDROXY GROUPS.

United States Patent C) U.S. Cl. 260-2105 15 laims ABSTRACT OF THE DISCLOSURE Cardioactive A -bufatrienolide rhamnoside ethers of the formula wherein R is methyl or formyl; R and R taken alone are hydrogen, lower alkyl, or acetyl; R and R taken together are /CHa CHa R is hydrogen, lower alkyl, or acetyl; and wherein at least one of R R or R is alkyl, and the method of preparing them by reaction of the corresponding 2',3',4- hydoxy compounds or 2,3-isopropylidene-4-hydroxy compounds with an alkyl halide, optionally followed by hydrolysis of the isopropylidene group and/or acetylation of unetherified hydroxy groups.

The present invention relates to A -bufatrienolide rhamnoside ethers and ether-acylates and to methods of making the same.

Many of the cardioglycosides important for the therapy of cardiac insufficiency in humans contain methylated sugar, e.g. digitalose (digitalinum verurn); oleandrose (oleanclrin); cymarose (strophanthoside, strophanthin, cymarin); and thevetose (thevetin, neriifolin, and peruvoside).

All of the glycosides mentioned have the methoxyl group in the 3-position of the sugar. However, glycosides are also known which have a methyl group on the 2'-hydroxy group, as are glycosides having a methyl group on each of the 2- and 3-hydroxy groups [cf. the summary of T. Reichstein et al. in Adv. Carboh. Chem. 17, 6S (1962)].

ice

It is of course known that the structure of the sugar or of the sugar chain has an influence on the elficacy of a cardioglycoside [B. Baumgarten, Die herzwirksamen Glykoside, VEB Georg Thieme-Verlag, Leipzig (1963), page 242]. However, heretofore it has not been possible to investigate quantitatively the influence of the position and number of ether groups present on the sugar on the pharmacological properties of cardioglycosides because insuflicient material for purposes of comparison was at hand.

It has already been attempted to prepare ethers from cardioglycosides by a partial synthesis. Makarevich describes the synthesis of cymarin from helveticoside [1. F. Makarevich, Med. Prom. SSSR, '29 1967)], as well as the synthesis of k-strophanthin-B from glucohelveticoside [1. F. Makarevich, Khim. Prirod. Soedin 2 17 (1967].

Dutch patent application 67.17502 further describes the preparation of helveticoside diethers from helveticoside or cymarin, and German Pat. 1,162,835 describes the preparation of the l9-rnethyl ether of 19-hydroxy cardenolides having an acyloxy group or glycoside group on the 3-carbon atom.

The references cited above teach either the formation of known cardioglycosides, or of fully etheri-fied cardioglycosides which have no free hydroxyl groups on the sugar, or of cardioglycosides which have an additional methyl group present on the aglycone but whose sugar portion remains unchanged. In no case are new compounds obtained having one or more additional ether groups on the sugar chain together with one or more free hydroxyl groups thereon.

The present invention relates more in particular to the preparation of new, defined, A -bufatrienolide rhamnoside ethers of the general formula wherein R is methyl or formyl; R and R taken separately are hydrogen, lower alkyl, or acetyl; R and R taken together form the group R, is hydrogen, acetyl, or lower alkyl; and wherein at least one of the groups R R or R is alkyl.

The present invention further relates more in particular to a process for the preparation of A -bufatrienolide rhamnoside ethers of the formula given above by reaction of a A -bufatrienolide rhamnoside derivative also be carried out as well as in inert organic solvents of the formula such as acetone, tetrahydrofuran, or dioxane, for example. For reaction, the reagents are suitably contacted at a temperature between about C. and 50 C. Most re- 5 actions are conveniently carried out at room temperature, 0 20 C. As is evident from the specific examples given I later herein, reaction times extending from a few minutes to several hours are used, depending on the specific reagents employed and the other conditions of the reaction.

Y Those skilled in the art will choose the shortest time giving good yields, as for any other chemical reaction.

R The assignment of structure to the resulting proscil- V I laridin and 19-oxo-pr0scillaridin monoethers is accom- 0H plished by nuclear magnetic resonance (NMR) spectro- HO O scopy, treatment with sodium periodate, and acetonide x1 formation. The assignment of structure to the resulting proscillaridin and l9-oxo-proscillaridin dimethylethers is H accomplished by specific syntheses from the pure monomethyl ethers and DC-comparison of the reaction prod- 20 ucts, as well as by NMR-spectroscopy. For all the proscilwhere! R 15 as earhef descnbidi R1 and R2 taken alone laridin ethers, the NMR-spectra of the acetylated ethers are hydrogen; or R and R taken together form the were also employed fer Structure: Studies group With the help of NMR-spectroscopy, proscillaridinand 19-oxo-proscillaridin-methyl ether mixtures can be analyzed. This requires both the NMR-spectra of the methyl ether mixture and, after acetylation of a small sample of a the mixture, of the acetates of the methyl ether mixture.

with an alkyl halide in an inert organic solvent in the For example, the composition of a proscillaridin-monopresence of a base. The product mixtures obtained can, methyl ether mixture can be determined in the following if desired, be separated by column chromatography and/ manner:

Area under the 4-OCH -signal 100 Area under all OGH -signals Percent content of 4t-methylether in the mixture:

Percent content of 3-methylcther in the mixture:

Percent content of 2-rnethylether in the mixturc=100% (percent content of 3- and 4-methylethers) The composition of a proscillaridin dimethylether mixture can be determined in the following manner:

Percent content of 2, 3-diinethylether in the mixture Area under the 4-O-acetyl signal in the dimethylether-acetate mixtureX 100 Total area under the O-acetyl signal in the dimethylether-acetate mixture Percent content of 3, 4-dimethylether in the mixture Area under the OCH -signal of the 3, 4-dimethylether-2-acetate in the dimethylether-acetate mixtureXlOO Total area under the OCH -signal in the dimethylether-acetate mixture Percent content of 2, 4-methylether in the mixture=l00% (percent content of 2, 3- and 3, 4'-dimethylethers) or by fractional crystallization and/or by counter-current The monomethylether mixtures and dimethylether mixdistribution. Further, any isopropylidene residue present tures can additionally be analyzed with the aid of preparain the compound may be split off and/ or free hydroxy tive thin layer chromatography, elution of the zones, and groups in the sugar residue may be acetylated if desired. quantitative determination With UV-spectroscopy. For

Alkyl iodides are particularly suitable as alkyl halides lTlPllomefhylethef i y mlxtufes, Cleavage for use in the reaction of the invention. The correspond- Wlth Sodium Periodate and tltfatlon 0f he Consumed reagent, or quantitative thin layer chromatography, can

also be used for determination of the 3'-ether. The data necessary for assignment of structure and for analysis of the mixtures can be taken from the following tables, in which Table I presents a summary of the R values, Table II is a review of the chemical properties, and Table III presents NMR data for several of the compounds of interest. In Table III the chemical dising bromides and chlorides, particularly the methyl and ethyl compounds, are not so easily obtainable as the iodides because of their greater volatility. However, the reaction also takes place with these compounds.

As bases for use in the reaction, alkaline earth hydroxides, mixtures of alkaline earth hydroxides and oxides, alkali metal hydrides and alcoholates such as sodi- 7 um hydride and sodium methylate, and silver oxide are placement, 5, i given in p.p.m. (s,,,,, ,,,,,,,=0.00 preferred. If the reaction is carried out with alkaline .m the couplings, J, are given in Herz. The following earth hydroxides or oxides, or with sodium hydride, dibb i i are d i T bl III; in 1et; d: methyl formamide is referably used as the solvent medidoublet; t=triplet; q=quadruplet; m=multiplet; br=

um. If silver oxide is used as the base, the reaction can broad.

TABLE Ia.R-VALUES OF PROSCILLARIDIN- AND 19- chlommmlethyl OXO-PROSCILLARIDIN- MONO- AND DI-DERIVATIVES. acetate, [ON SILICA GEL (HF-ME ROK) WITHOUT CHAMBER SATURATION] Compound 4 1 2 1 1 1 Chlorofonn/acetone 5 Ethyl Prosc1l1aridin-2,3-acetonide-4-ethylether 0.31 0.48 0.71 Compound acetate 4/1 2/1 1/1 lgrosclllarlgln-gi-mezgylefEer-liZAi-fiiacfifle- 0. 0. 0. 51 rosci ari in- -me ye er-2,4- acetate..." 0.15 0.26 0.54 Proscillaridin-2-methylether. 0. 28 0. 10 0. 26 0. 47 Proscillaridin-4-methylethcr-2,3-diacetate. 0. l6 0. 28 0. 55 Proscillaridin-3-methylethe 0. 30 0. 10 0. 27 0. 51 Proscillarldin-2,3-dimethylether-4acetate 0. 11 0. 19 0. 38 Prosci1lar1din-4-methy1ethe 0.37 0.10 0.28 0. 53 Proscillarid111-2,4-dimethylether-3-acetate-- 0.17 0.25 0.50 19-oxo-proscillarldin-3-methyl he 0.23 0.07 0.18 0.41 Proscillaridin-3,4-djmethylether-2-acetate 0.15 0.24 0.49 1Q-oxo-proscillaridin-4-methylether 0.27 0. 07 0.18 0.43 10 Proscillaridin-3'-ethylether-2,4-diacetate 0.22 0. 0.57 Proscillar1dln-3-ethylether 0.47 0. 17 0.40 0. 67 Proscillaridin-4-ethylether-2,3-diacetate 0. 22 0. 35 0. 56 Prosclllarldin-4-ethylether 0.48 0. 15 0.35 0. Proscifiarig%n2:,i;-gimegyleer. 0. 47 0. 22 0. 5g 0. I Y @1119 Ye 5 TABLE Ila-CHEMICAL PROPERTIES OF PROSCILLARI- g ggg g ygmg 79 DIN- AND 19-02mm ROSCILLARIDIN-MONOETHE RS etneln... 0.34 0 2o 0. 42 0. 66 15 Acetonide tfig r i ffj ff 0 41 0 22 0 46 0 6e Compmmd cleavage mmtm 19-oxo-proscillarldin-3,4-dimethyl- 11 ether 0.41 0 22 0.46 0 69 1 i3ZZ513335353333 112?I i PT059i11aTidin'3methY1eth9T-4" Proscillarldin4-methylether: acetate 69 0 38 63 0 81 19-oxo-prosclllarldin-3-methylether 19-ox0-pr0scillaridin-4-methylether 20 Proscillaridin-3-ethylether Proscillaridin-4-ethylether TABLE IIb.S'IRUCTURAL ASSIGNMENT OF PROSCILLA- RIDIN-DIMETHYLETHERS BY PARTIAL METHYLATION TABLE Ib.R:-VALUES OF PROSCILLARIDIN- AND'lQ-OXO- 2 OF PROSCILLARIDIN-MONOMETHYLETHERS PROSCILLARIDIN-TRI-DERIVATIVES. [ON SILICA GEL 5 (HF-ME RCK) WITHOUT CHAMBER SATURATION] Obtained by pafrtial methylation chloroform/ethyl acetate- Proscil- Proscil- Prosclllaridlnlarldinlarldln- Compound 4/1 2/1 1/1 2-methyl- 3-methyl- 4-methyl- 3Q Dimethylether ether ether ether Prosc1llar1d1n-2,3,4-trin1ethylether 0. 13 0.21 0.40

19-oxo-proscillaridln-2,3,4-trimethylether 0.06 0.10 0. 23 Proscillaridin-2,3-dimethylether Proscillarldln-2,3-acetonide-4-methylether.... 0.28 0.43 0. 67 Proscillarldin-2,4-dhnethylether 19-oxo-proscillarld1n-2,3-acetonide-4- Prosclllarldin-3,4-dimethylether.-

methylether 0.06 0.10 0. 25

TABLE IIIa.-NMR DATA FOR PROSCILLARIDIN-METHYLETHE RS Proscillaridin- Proscillan'din- Proscillarldin- Proscillaridln- 2-methylether 3-methylether 4-methylether 2 ,3 -dimethylether 5.00 (d;J=1.5).................. 4.97 (s)...................... 5.10 (d;.T=1). (complex system) 4.07 (q; =1, 3.3-4.0 (complex system). 3.4-4.0 (complex system). (1 3.3-4.0 (complex system) ..do Do. ..do .09 (br.t,J1=.Tz=910) Do. do 4.0 (not assignable) Do.

H-fi 1.31 (d; J 2 ((1; (i=6) 1.31 (d; J=5.5).

H-19 1.07 (s)..... 1.06(s).

-OCH 3.53 (s) 3.50 (S)... 3.50 and 3.54 (s).

Proscillaridin- Proscillaridin- Proscillaticlin- Proscillarldln- 2,4-dimethy1ether 3,4'-dimethylether 2 ,3 ,4-tnmcthylether 2',3-acctonide-4-methy1ether 5.02 (d; J=1) 5.07 (br. s) 5.18 (sharp s).

4.06 (q; 1 :1, Jz=3) 3.4-3.8 (complex system) 4.15-4.22 (AB-system). 3.4-4.0 (not assignable) do Do.

3.09 (t; L: 3.15 (t; J1=Jn=910)... ca. 3.0 (complex multiplet). 3.4-3.9 (not assignable). 3.4-3.8 (not assignable).... 3.5-3.9 (m.)

1.30 (d; J=6.5) 1.29 (d; J=6) 1.27 (d; J=6).

1.05 (s) (s) 1.07 (s).

3.53 and 3 58 (s) 3.51, 3.54 and 3.58 (s) 3.56 (s).

TABLE IIIb.-NMR DATA FOR 19-OX0-PROSCILLARIDIN-METHYLETHERS 19-oxo-proscillarldln- 19-oxo-proscillaridin- 19-ox0-proscil1aridin- 19-oxo-prosci1laridin- 3-methy1ether 4-methylether 2, 3-dimethylethor 2,4-dimethylether 4.93 (bns) 5.06 (br. s) 5.0 (br.). 3.3 %0 (complex system) 33-411) (complex system) 34-4150 (complex system).

o o o.

a. .44.0 (not assignable).

5.04 (br.) 5.16 (sharp s).

omplex system) 4.1-4.2 (AB-system).

5.0 (br.) 3.4-4.0 (complex system) d H-4 ca 3.0 3.0 (m).

4-4.0 (not assignable). 3.7 (m.).

H-6' 1 29 (d; =6) 1.25 (d; J=6).

0.72 (s)... 0.72 (s). H-19 9.82 (s) .80 (S 9.82 (s). -OCH 8.50 and 3.56 (s) 3. 3.57 (s).

TABLE IIIC.-NMR DATA FOR PROSCILLARIDIN-METHYLETHER-ACETATES Proscillaridin-2-n1ethylether- Proscil1aridin-3'-methylether- Prosei1laridin-3-methylether- Prsclllaridin-4'-methylether- 3, 4-diaeetate 4-acetate 2, 4-diacetate 2, 3-diacetate 1) 6.06 (br. s).

4.11 5.37 (AB-system). 3.57 Do.

504 Oar. t; J1=J 3.90 4.1 (not; assignable). 1.17 (d; J=6.5).

2.09 and 2.15 (s) Proseillan'dln-3', 4'-dimethylether-2- Proseillarldin-Z',3-dimethylether-4-aeetate Proscillaridin-2,4-dimethylether-3-acetate acetate H-l 5.08 (s) 5.00 (br H2' .43.8 (not assignable) H- 3.95 (q; J =4, J2=1o 5.22 (q, H4;. 5.07

TABLE IIId.NMR DATA FOR PROSCILLARlDIN-ETHYLETHE RS Proscillaridin-3-ethylether Proseillaridin-4ethylether Proseillaridin-2,3-acetonide- 4-ethylether 17 b'. 3.54.0 (not assignable). 1.30 (d; .T=6.5)

5.18 (sharp s). 4.15-5.22 (AB-system). 0.

TABLE IIIe.-NMR DATA FOR PROSOILLARIDIN-ETHYL- ETHER-ACETATES Proseillarldin-Imethyl- Proscillaridin-i -ethy1- ether-2,4'-diaeetate ether-2,8-diacetate H-l 4.99 (d; J=1) 4.89 (br. s). H-2' 5.29 (q; J1=1, 12:3) H-3 3.4-4.0 (m; not assignable). 5.20-5.36 (AB-system).

o. H-4 5.00 (t; J1=Jz=910) 3.28 (br. t; I =Jz=9-l0). H-5 3.44.0 (nota assignable). 11-6- 1.33 (d; J'=6). 11-18-- 0.74 (S). H-l9 1.05 (S). -OC:H5. 3.68 (q; J=7).

OAc 2 2.15 (s) 2.05 and 2.15 (s).

The new compounds possess very good cardioactivity and are readily resorbable. Following Table TV shows the superior effect of the new compounds in comparison with proscillaridin.

The determination of cardioactivity in guinea pigs is according to Knafll-Lenz, J. Pharm. exp. Ther. 29, 407 (1926) and that in cats is according to Hatcher et al., Am. J. Pharm. 82, 360 (1910). Enteral resorption is cats is determined according to R. Hotovy, Arzneimittelforsch g 160 (1 The new compounds can be administered orally in pharmaceutically conventional forms such as tablets, dragees, capsules, solutions, and the like. The dosage range is between 0.2 and 3.0 mg. per patient per day.

A better understanding of the present invention and of its many advantages will be' had by referring to the following specific examples given by way of illustration.

EXAMPLE 1 5 g. of proscillaridin were stirred for 6 hours at 20 C. with 20 ml. of methyl iodide and 10 g. of silver oxide in ml. of dioxane. The batch was filtered, the residue washed thoroughly with ethyl acetate, the filtrate evaporated, and the residue chromatographed on inactivated silica gel -in a column X 5 cm. with the system chloroform/acetone=2/ 1. The following were obtained:

0.58 g. of proscillaridin-dimethylether mixture 11% of theory), R value=0.47-0.59 in ethyl acetate;

2.76 g. of proscillaridin-monomethylether mixture (54 percent of theory), 'R -value=0.28-0.37 in ethyl acetate; and

1.22 g. of proscillaridin (24,.% of the starting material).

Crystallization of 2.76 g. of proscillaridin-monomethyl ether mixture from 25 ml. of ethyl acetate gave 1.29 g. of crystalline proscillaridin-3'-methyl ether, M.P. 235-242 C.; R,-value=0.30 in ethyl acetate.

EXAMPLE 2 5 g. of proscillaridin were stirred for 2 hours at 20 C. with 30 g. of silver oxide and 30 ml. of methyl iodide in 100 ml. of dirnethylformamide. The batch was filtered, the residue thoroughly washed with ethyl acetate, and the filtrate evaporated. After column chromatography on inactivated silica gel using a column 1100 X 6 cm. with ethyl acetate, the following was obtained:

1.30 g. of proscillaridin-monomethylether mixture (25% of theory), R -value=0.28-0.37 in ethyl acetate.

9 EXAMPLE 3 g. of proscillaridin were heated briefly to boiling in 50 ml. of acetone with 1 g. of boric acid. Thereafter, the clear solution Was cooled to 40 C., combined with 20 ml. of methyl iodide and g. of silver oxide, and stirred for 16 hours at 40 C. The batch Was filtered, the residue thoroughly Washed with ethyl acetate, and the filtrate evaporated. Column chromatography on inactivated silica gel on a column 90 X 5 cm. gave:

0.84 g. of proscillaridin-dimethylether mixture (16% of theory), R -value=0.470.59 in ethyl acetate; and

3.04 g. of proscillaridin-monomethylether mixture (59% of theory), R -value=0.28-0.37 in ethyl acetate.

After crystallization from 30 ml. of ethyl acetate, 1.84 g. of crystalline proscillaridin-3'-methyl ether are obtained (36 percent of theory), Ri-value=O.30 in ethyl acetate; M.P.=231-240 C.

EXAMPLE 4 5 g. of proscillaridin were stirred for 6 hours at 20 C. in 100 ml. of tetrahydrofuran with 20 ml. of ethyl iodide and 10 g. of silver oxide. The batch was worked up as described in Example 1.

After column chromatography on inactivated silica gel using a column 110 x 5 cm. and the system chloroform/ acetone-=4/ 1, the following are obtained: 2.26 g. of proscillaridin-monoethylether mixture (43% of theory, or 90% of theory calculated on the reacted starting material), Rf value=0.47 in ethyl acetate; and 2.63 g. of proscillaridan (53 percent of the starting material).

Crystallization of the proscillaridin-monoethylether mixture from 10 ml. of ethyl acetate gave 0.77 g. of proscillaridin-3'-ethyl ether percent of theory, or 31 percent of theory calculated on the reacted starting material), R value==0.47 in ethyl acetate; M.P.=238-249 C. 249 C.

EXAMPLE 5 5 g. of proscillaridin were stirred at C. for 70 minutes with 33 ml. of methyl iodide and 2.5 g. of finely-powdered barium hydroxide in 100 ml. of dimethylformamide. The batch was taken up into 500 ml. ethyl acetate, shaken twice with 5 percent aqueous HCl, then twice with 5 percent aqueous sodium hydroxide, and, finely, twice with water. The organic phase was dried over anhydrous sodium sulfate and evaporated.

After column chromatography on inactivated silica gel using a column 80 x 5 cm. and the system chloroform/ acetone==3/ 1, the following are obtained: 0.98 g. of proscillaridin-dimethylether mixture (19% of theory), R -value=0.47-0.59 in ethyl acetate; and 3.46 g. of proscillaridin-monomethylether mixture (67 percent of theory), R,-value=0.28-0.37 in ethyl acetate.

Crystallization of the proscillaridin-monoethylether mixture from 35 ml. of ethyl acetate gave 2.23 g. of crystalline proscillaridin-3-methyl ether (44 percent of theory, R;-value=0.30 in ethyl acetate; M.P.=248 -255 C.

EXAMPLE 6 5 g. of proscillaridin were stirred in 50 ml. of dimethylformamide with 5 ml. of methyl iodide and 3.5 g. of finely-powdered barium hydroxide for 1 hour at 20 C. The product was further worked up as described in Example 5. After column chromatography the following were obtained:

1.61 g. of proscillaridin-dimethylether mixture (31 percent of theory), R -value=0.47-0.59 in ethyl acetate; and

2.34 g. of proscillaridin-monomethylether mixture (46 percent of theory), R -value=0.28-0.37 in ethyl acetate.

Crystallization of the proscillaridin monomethylether mixture from ml. of ethyl acetate gave 1.68 g. of proscillaridin-3-methyl ether (33 percent of theory), R;- value=0.30 in ethyl acetate; M.P.=233-240 C.

10 EXAMPLE 7 5 g. of proscillaridin were briefly heated to boiling in ml. of dimethylformamide with 33 m1. of methyl iodide, 33 g. of finely-powered barium oxide, and 1.4 g. of finely-powdered barium hydroxide, and then stirred for a further two hours at 20 C. The product was worked up as described in Example 5.

After column chromatography the following were obtained:

1.27 g. of a non-polar fraction which comprises proscillaridin-triand di-methyl ethers, as well as other nonpolar by-products; and

1.35 g. of proscillaridin-monomethylether mixture (26 percent of theory), R -value=0.28-0.37 in ethyl acetate.

Crystallization of the proscillaridin-monomethylether mixture from 50 ml. of ethyl acetate gave 0.77 g. of crystalline proscillaridin-3-methylether (15% of theory), R value=0.30 in ethyl acetate; M.P.-=233-239.5 C.

EXAMPLE 8 2.5 g. of finely-powdered barium hydroxide were stirred for 16 hours at 20 C. in 100 ml. dimethylformamide. 5 g. of proscillaridin and 33 ml. of methyl iodide were then added and the batch was stirred for another 60 minutes at 20 C. Further working up followed as described in Example 5.

After column chromatography the following were obtained:

0.36 g. of proscillaridin-dimethylether mixture (7 percent of theory), R -value=0.47-0.59 in ethyl acetate; and 2.79 g. of proscillaridin-rnonomethylether mixture (54 percent of theory), R,-value=0.28-0.37 in ethyl acetate.

Crystallization of the proscillaridin monomethylether mixture from 30 ml. of ethyl acetate gave 1.5 g. of crystalline proscillaridin-3'-methyl ether (31 percent of theory), R -value=O.30 in ethyl acetate; M.P.=234240 C.

EXAMPLE 9 3 g. of finely-powered barium hydroxide were stirred for 16 hours in 50 ml. of dimethylformamide at 20 C. 5 g. of proscillaridin and 5 ml. of methyl iodide were then added and the batch was stirred for a further 45 minutes at 20 C. The material was then Worked up as described in Example 5.

After column chromatography, the following are obtained:

1.06 g. of proscillaridin-dimethylether mixture (20 percent of theory), R -value=0.470.59 in ethyl acetate; and

3.91 g. of proscillaridin-monomethylether mixture (76 percent of theory), R -value=0.28-0.37 in ethyl acetate.

Crystallization of the proscillaridin-monomethylether mixture from 40 ml. of ethyl acetate gave 2.75 g. of

crystalline proscillaridin-3'-methylether (54% of theory),

R -value=0.30 in ethyl acetate; M.P.=246252 C.

Ten further batches prepared analogously with reaction times of from 20 to 30 minutes gave from 0.79 g. to 1.39 g. of dimethylether mixture (15-27 percent of theory) and 3.33 to 4.01 g. of proscillaridin-monomethylether mixture (65-78 percent of theory), and, after crystallization from ethyl acetate, 2.40 to 2.99 g. of crystalline proscillaridin-3'-methyl ether (47-58 percent of theory).

A 19-oxo-pro'scillaridin-monomethylether mixture having an R -value of 0.23-0.27 in ethyl acetate is obtained in analogous fashion. After crystallization from ethyl acetate, pure 19-oxo-proscillaridin-3'-methyl ether is obtained, M.P.=226-230 C., R -value=0.23 in ethyl acetate.

EXAMPLE 10 9.91 g. of proscillaridin-monomethylether mixture (from the mother liquor remaining after the crystalliza- 1 1 tion of proscillaridin-3'-methyl ether in Example 9) were subjected to countercurrent distribution in a system involving 100 tubes and 2000 transfers using the system carbon tetrachloride/ chloroform/ methanol/ water: 2/ 2/ 3/1 (V:25/25 ml.; T=20 C.).

After 100 transfers in the basic process, a process involving alternate phase removal was employed in which the ratio of the removed upper phases to the lower phases was about 3:2 (in total, 1234 upper phase transfers and 766 lower phase transfers).

The following were obtained in the tubes:

-38: 1.21 g. of a mixture comprising proscillaridin-2'- and -4'-methyl ethers, R;-value:0.28 and 0.37 in ethyl acetate;

39-74: 1.37 g. of proscillaridin- -methylether, R -value: 0.28 in ethyl acetate; crystals from ethyl acetate, M.P.:151.5-158.5 C.;

75-100: 0.52 g. of a mixture of proscillaridin-2'- and -3'-methyl ethers, R -value:'0.280.39 in ethyl acetate.

The discarded lower and upper phases contained proscillaridin-4'- and -3'-rnethyl ethers.

EXAMPLE l1 5 g. of proscillaridin were stirred for one hour at 20 C. in 100 ml. of dimethylformamide with 25 ml. of ethyl iodide and a suspension of about 50 percent sodium hydride in oil. The batch was combined with 500 ml. of ethyl acetate, shaken three times with 500 ml. portions of water, with each aqueous phase being washed again with 500 ml. of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, evaporated, and the residue chromatographed on inactivated silica gel with a column 100 x 5 cm. using the system chloroform/ acetone:4/1-1/1.

The following were obtained:

0.48 g. of proscillaridin-diethylether mixture, R -value:

0.5 in chloroform/acetone:4/ 1;

2.17 g. of proscillaridin-monoethylether mixture, R -value :0.47 in ethyl acetate; and

1.77 g. of proscillaridin (35% of the unchanged starting material).

With 1.0 g. of a 50 percent sodium hydride-oil suspension, instead of 0.5 g., the following are obtained under otherwise identical conditions:

0.30 g. of proscillaridin-triethyl ether, R;-value:0.9 in

chloroform/-acetone:4/ l;

1.06 g. of proscillaridin-diethylether mixture;

2.82 g. of proscillaridin-monoethylether mixture; and

0.24 g. of proscillaridin (5 percent of the unchanged starting material).

In an analogus fashion, proscillaridin monopropylether mixtures, R;-value:0.55 in ethyl acetate, and proscillaridin monopentylether mixtures, R -va1ue:0.60 in ethyl acetate, are obtained in small yields from proscillaridin and propyl iodide and pentyl iodide respectively.

In the methylation of proscillaridin, the sodium hydride-oil suspension can also be replaced 'by sodium methylate. The yields of proscillaridin monomethylether mixture are, however, smaller than with sodium hydride.

EMMPLE 12 2.5 g. of proscillaridin-2',3'-acetonide were stirred for 20 hours at 20 C. in 50 ml. of dimethylformamide with 15 ml. of methyl iodide and 15 g. of silver oxide. The material was further worked up as described in Example 2. After column chromatography on inactivated silica gel on a column 100 x 4 cm. with hte system chloroform/ethyl acetate: 1, the following are obtained:

1.90 g. of proscillaridin-2',3'-acetonide-4'-methyl ether (74 percent of theory), amorphous, R -va1ue:0.67 in chloroform/ ethyl acetate: 1/ l.

12 EXAMPLE 13 10 g. of proscillaridin-2',3'-acetonide were boiled for 6 hours in 200 ml. of acetone with 200 ml. of ethyl iodide and 50 g. of silver oxide. The material was worked up further as described in Example 1.

After column chromatography on inactivated silica gel on a column 140 x 3 cm. with the system chloroform/ ethyl acetate:4/1-1/1, the following are obtained:

1.63 g. of proscillaridin-Z',3'-acetonide-4-ethyl ether (66 percent of theory calculated on the reacted starting material), amorphous, R;-value:0.7l in chloroform/ ethyl acetate: 1/ 1; and

7.64 g. of proscillaridin-Z', -acetonide recovered unchanged.

EXAMPLE 14 1 g. of proscillaridin-2,3-acetonide-4'-ethyl ether was stirred in 50 ml. of 0.2 N hydrochloric acid in tetrahydrofuran for 8 hours at 20 C. The batch was taken up into 200 ml. of ethyl acetate, the organic phase was shaken once with 200 ml. of 5 percent aqueous sodium hydroxide and twice with water, dried over anhydrous sodium sulfate, and evaporated. The residue was chromatographed on inactivated silica gel in a column x 3 cm. with the system chloroform/ethyl acetate:4/ l.

The following are obtained:

0.14 g. of proscillaridin 2',3 acetonide-4-ethyl ether (:14 percent unconverted starting material), R;- value:0.71 in chloroform/ethyl acetate:l/ 1 and 0.46 g. of proscillaridin-4-ethyl ether (48 percent of theory), R -value:0.48 in ethyl acetate; crystals from ethyl acetate, M.P.:l82-192 C.

EXAMPLE 15 5 g. of proscillaridin-2',3'-acetonide were dissolved in 50 ml. of dimethylformamide and stirred with 10 ml. of methyl iodide and 1 g. of a 50 percent sodium hydrideoil suspension for 15 minutes at 20 C. After further working up as in Example 10 and column chromatography on inactivated silica gel with a column x 5 cm. using the system chloroform/ethyl acetate:5/ 1, are obtained:

3.83 g. of proscillaridin-Z',3-acetonide-4-methyl ether (75 percent of theory), amorphous, R -value:0.67 in chloroform/ ethyl acetate: 1 1.

EXAMPLE l6 5 g. of proscillaridin-2,3-'acetonide were dissolved in 50 ml. of dirnethylformamide and stirred with 10 ml. of ethyl iodide and 1 g. of a 50 percent sodium hydride-oil suspension for 15 minutes at 20 C. After further workng up as in Example 10 and column chromatography on inactivated silica gel in a column x 5 cm. using the system chloroform/ethyl acetate:5/ 1, the following are obtained.

1.50 g. of proscillaridin-2',3'-acetonide-4'-ethyl ether (29 percent of theory), amorphous, R -value:0.71 in chloroform/ ethyl acetate: 1/ 1.

yl iodide, and 6 g. of finely-powdered barium hydroxide, were stirred for 3 hours at 20 C.

The batches were combined and further worked up according to Example 5. The residue on evaporation was chromatographed on inactivated silica gel on a column x 6 cm. with the system. chloroform/ethyl acetate=4/ 1. Recovered starting material was resubmitted to methylation according to the preceding description, and the starting material recovered was once more methylated according to the procedure given. After three methylations and three column chromatographs, the following materials are obtained, in total: 26.82 g. of proscillaridin- 2,3-acetonide-4'-methyl ether (65 percent of theory or 76 percent of theory calculated on the reacted starting material), amorphous, R -value=0.67 in chloroform/ ethyl acetate=1/1; and 5.64 g. of proscillaridin-2',3-acetonide (14 percent of the starting material).

In an analogous fashion, 19-oxo-proscillaridin-2',3'-acetonide-4'-methyl ether is obtained, amorphous, R -value=0.25 in chloroform/ethyl acetate=1/ 1.

EXAMPLE 18 25 g. of proscillaridin-2',3'-acetonide-4'-methyl ether were stirred for 18 hours at 20 C. in 1.25 l. of 0.2 N hydrochloric acid in tetrahydrofuran. The batch was further worked up as described in Example 14 and the residue obtained on evaporation was chromatographed on inactivated silica gel with a column i160 x 6 cm. with the system chloroform/ethyl acetate=1/1-pure ethyl acetate. The following are obtained: 8.93 g. of proscillaridin-4'-methylether (39 percent of theory), R -value=0.37 in ethyl acetate, crystals from ethyl acetate, M.P. 197204.5 C.

EXAMPLE 19 1.8 g. of 19-oxo-proscillaridin-2',3'-acetonide-4'-rnethyl ether were stirred for 6 hours at 20 C. in 90 ml. of 0.2 N hydrochloric acid in tetrahydrofuran. The batch was further worked up as described in Example 14 and the residue obtained on evaporation was chromatographed on inactivated silica gel on a column 100 x 4 cm. with ethyl acetate. In this fashion are obtained:

0.37 g. of 19-oxo-proscillaridin-2,3'-acetonide-4-methyl ether (E21 percent of the starting material employed), R -value=0.25 in chloroform/ethyl acetate=1/l; and

0.96 g. of 19-oxo-proscillaridin-4'-methyl ether (57 percent of theory), R -value=0.27 in ethyl acetate, crystals from ethyl acetate, M.P.=222229 C.

EXAMPLE 20 g. of proscillaridin were dissolved in 100 ml. of dimethylformamide and stirred for one hour at 20 C. with 20 ml. of methyl iodide and 2.0 g. of an about 50 percent sodium hydride-oil suspension. After further working up as in Example 10, followed by column chromatography on inactivated silica gel with a column 120 x 4 cm. using the system chloroform/ethyl ester=2/ 1, the following were obtained:

1.74 g. of proscillaridin-trimethyl ether (32 percent of theory), R -value=0.40 in chloroform ethyl acetate= 1/ 1, crystals from methanol, M.P.: 121 124 C.;

2.19 of proscillaridin-dimethylether mixture (42 percent of theory), R -va1ue=0.47-0.59 in ethyl acetate; and 0.37 g. of proscillaridin-mo-nomethylether mixture (7 percent of theory), R;-value=0.28-0.37 in ethyl acetate.

EXAMPLE 21 10 g. of proscillaridin were stirred for 3 hours at 20 C. in 200 ml. dimethylformamide with 60 ml. of methyl iodide and 12 g. of finely-powdered barium hydroxide. The batch was further worked up as described in Example 5, and the evaporation residue was chro-matographed on inactivated silica gel in a column 120 x 5 cm. with the system chloroform/ethyl acetate:4/ 1.

In this manner are obtained:

3.02- g. of proscillaridin-2',3',4'-trimethyl ether (28 percent of theory), R -value=0.40 in chloroform/ethyl acetate=1/ 1, crystals from methanol, M.P.=119- 123 C.; and

6.15 g. of proscillaridin-dimethylether mixture (58 percent of theory), after thin layer chromatography, approximately 90 percent of proscillaridin-2,3-dimethyl ether is obtained, R -value=0.47-0.59 in ethyl acetate.

A further methylation of the diethylether mixture according to the procedure described above, followed by chromatography on a column 120 x 5 cm. with the system chloroform/ethyl acetate=4/1 produces the further products:

1.98 g. of proscillaridin-2,3,4-trimethyl ether (total=46 percent of theory), R -value=0.40 in chloroform/ethyl acetate=1/ 1; and

3.36 g. of proscillaridin-2',3-dimethyl ether (32 percent of theory), R -value=0.47 in ethyl acetate, crystals from a small amount of ethyl acetate, M.P.=136- 140.5 C.

19-oxo-proscillaridin 2',3',4' trimethyl ether, amorphous, R -value=0.23 in chloroform/ethyl acetate=1/ 1, and 19-oxo-proscillaridin-2',3-dimethyl ether, amorphous, R -value=0.34 in ethyl acetate, are obtained in an analogous manner.

EXAMPLE 22 5 g. of proscillaridin-4-methy1 ether were stirred for minutes at 20 C. in 50 ml. of dimethylformamide with 5 ml. of methyl iodide and 2 g. of finely-powdered barium hydroxide. The product was further worked up as described in Example 5. After column chromatography on inactivated silica gel using a column .100 x 5 cm. and the system chloroform/ethyl acetate=1/ 1, the following products are obtained:

0.90 g. of proscillaridin-2,3',4'-trimethyl ether (17 percent of theory), R -value=0.40 in chloroform/ethyl acetate=1/1, crystals from methanol, M.P.=124- 129 C.;

2.68 g. of a mixture of proscillaridin-2',4'- and -3',4-dimethyl ethers (51 percent of theory), R -value=0.55- 0.59 in ethyl acetate; and

0.23 g. of proscillaridin-4'-methyl ether (E5 percent of the starting product), R -value=0.37 in ethyl acetate.

In an analogous fashion, starting from 19-oxo-proscillaridin-4'-methyl ether, a mixture of 19-oxo-proscillaridin- 2,4'- and -3',4'-dimethyl ethers is obtained, R -value= 0.41 in ethyl acetate.

From proscillaridin-2'-methyl ether, a mixture of proscillaridin 2,3- and -2',4' dimethyl ethers, R value=0.47 and 0.55 in ethyl acetate, is obtained. After thin layer chromatography, the mixture comprises about percent of proscillaridin-2',3'-dimethyl ether and about 10 percent of proscillaridin-2',4'-dimethyl ether. The mixture can be separated into its pure components by column chromatography.

In the same manner, a mixture of proscillaridin-2',3'- and -3',4'-dimethyl ethers, R -value=0.47 and 0.59 in ethyl acetate, is obtained from proscillaridin-3'-methyl ether. After thin layer chromatography, the mixture comprises about 90 percent of proscillaridin-2',3'-dimethyl ether and about 10 percent of proscillaridin-3,4-dimethyl ether. The mixture can be separated into its pure components by column chromatography.

EXAMPLE 23 2.68 g. of a mixture of proscillaridin-2',4- and -3,4- dimethyl ethers (obtained according to Example 20) were subjected to countercurrent distribution in tubes for 550 transfers using the system carbon tetrachloride/ chloroform/methanol/water=3/1/3/1 (V=25/25 ml.; T=20 C.). After 100 steps in the basic process and 50 steps removing the upper phase, a process for alternate phase removal was employed with the relationship between the removal upper phase to the lower phase of about 13:7 (in total 405 upper phase transfers and 15 lower phase transfers). The following materials are obtained in the tubes:

18-47:0.89 g. of proscillaridin-3',4'-dimethy1 ether,

R -value:0.59 in ethyl acetate, crystals from ethyl acetate, M.P.:218.5-223 C.;

48-63:0.8O g. of a mixture of proscillaridin-2',4'- and -3',4'-dimethyl ethers, R -value:0.55 and 0.59 in ethyl acetate;

64-99:O.82 g. of proscillaridin-2,4-dimethyl ether,

R -value=0.55 in ethyl acetate, crystals from ethyl acetate, M.P.:2l0.5-214 C. A mixture of 19-oxo-proscillaridin-2',4- and -3,4'-

dimethyl others can be separated into its pure components in a similar manner.

EXAMPLE 24 200 mg. of proscillaridin-4'-methyl ether were combined in ml. of absolute pyridine with 5 ml. of acetic anhydride and left to stand for 24 hours at 20 C. ml. of methanol were than added to the reaction mixture to destroy excess acetic anhydride. After 1 hour at C., the mixture was taken up with 200 ml. of ethyl acetate, the organic phase extracted twice with 200 ml. portions of 5 percent aqueous HCl, twice with 200 ml. portions of 5 percent aqueous NaOH, and twice with 200 ml. portions of water, dried over anhydrous sodium sulfate, and evaporated. Column chromatography of the evaporation residue on inactivated silica gel using a column 60 x 2 cm. with the system chloroform/ethyl acetate:4/l produces:

202 mg. of proscillaridin-4'-methyl ether-2,3'-diacetate (88 percent of theory), amorphous, R;-value:0.55 in chloroform/ethyl acetate: 1/ 1.

The following products are synthesized by identical procedures from the indicated starting materials:

Proscillaridin-2'-methyl ether: Proscillaridin-2 methyl ether 3',4 diacetate, amorphous, R -value:0.51 in chloroform/ ethyl acetate: 1 1;

Proscillaridin 3'-methyl ether: Proscillaridin-3'-methyl ether 2',4-diacetate, amorphous, R -value:0.59 in chloroform/ ethyl acetate: 1 1;

Proscillaridin 2,3'-dimethyl ether: Proscillaridin- 2',3'-

dimethyl ether-4-acetate, amorphous, R -value:0.38 in chloroform/ethyl acetate:1/ 1;

Proscillaridin 2',4-dimethyl ether: Proscillaridin-2',4'-

dimethyl ether-3-acetate, amorphous, R -value:0.50 in chloroform/ ethyl acetate: 1 1;

Proscillaridin 3,4'-dimethyl ether: Proscillaridin-3',4'-

dimethyl ether-2-acetate, amorphous, R -value=0.49 in chloroform/ ethyl acetate:l/ 1;

Proscillaridin-3-ethyl ether: Prosci1laridin-3'-ethyl ether- 2',4'-diacetate, amorphous, R -value':O.S7 in chloroform/ethyl acetate:1/ 1; and

Proscillaridin-4-ethyl ether: Proscillaridin-4'-ethyl ether- 2,3'-diacetate, amorphous, R -va1ue:0.56 in chloroform/ ethyl acetate: 1/ 1.

In an analogous fashion, 19-oxo-proscillaridin-methyl ether-acetates can be prepared from the corresponding 19- oxo-proscillaridin-methyl ethers.

EXAMPLE 1 g. of proscillaridimdimethyl ether mixture (comprising about 80 percent proscillaridin-2',3'-dimethyl ether) were combined in 10 ml. absolute pyridine with 10 ml. of acetic anhydride and left to stand for 24 hours at 20 C. The material was further worked up as described in Example 24. After column chromatography on inactivated silica gel using a column 120 x 3 cm. and the system chloroform/ethyl acetate:4/ 1, the following are obtained:

0.12 g. of a mixture of prosci1laridin-2,4'-dimethyl ether- 3'-acetate and -3,4'-dimethyl ether-2-acetate (11 per- 16 cent of theory), R -value:0.50 in chloroform/ethyl acetate:l/1; and 0.73 g. of proscillaridin-2,3-dimethyl ether-4'-acetate (68 percent of theory), R -value:0.38 in chloroform/ ethyl acetate:1/ 1.

EXAMPLE 26 10 g. of proscillaridin-3-methyl ether were combined in ml. of absolute pyridine with 10 ml. of acetic anhydride and left to stand for two hours at 20 C. The material was further worked up as described in Example 24. After column chromatography on inactivated silica gel using a column x 5 cm. and the system chloroform/acetone:3/ 1, the following are obtained:

1.62 g. of proscillaridin 3 methyl ether-2',4'-diacetate (14 percent of theory), amorphous, R;-value:0.54 in chloroform/ ethyl acetate: 1/ 1;

0.36 g. of proscillaridin-3-methyl ether-2'-acetate (3 percent of theory), amorphous, R -va1ue:0.72 in ethyl acetate;

5.39 g. of proscil1aridin-3-methyl ether-4-acetate (50 percent of theory), amorphous, R -value:0.69 in ethyl acetate;

2.92 g. of proscillaridin-3'-methyl ether (:29 percent of recovered starting material), R -value:0.30 in ethyl acetate.

Using an analogous procedure, a mixture of proscillaridin-2'-methyl ether-3-acetate and -2'-methyl ether- 4'-acetate, R -value:0.700.72 in ethyl acetate, is obtained from proscillaridin-2'-methyl ether; a mixture of proscillaridin-4-methyl ether-2'-acetate and -4'-methyl ether-3-acetate, both having an R -value:0.72 in ethyl acetate, is obtained from proscillaridin-4'-rnethyl ether.

What is claimed is:

1. A A -bufatrienolide rhamnoside ether of the formula 18 14. A process for the preparation of a A -bufawherein R has its earlier meaning, with a lower alkyl trienolide rhamnoside ether of the formula halide in an inert organic solvent in the presence of a base, and then hydrolytically cleaving the isopropylidene group present in the resulting A -bufadienolide 5 rhamoside ether by contact with a mineral acid.

0 15. A process as in claim 14 wherein hydroxy groups i I in said A -bufatrien0lide rhamnoside ether are subsequently acetylated by contact with acetic anhydride in I the presence of an acid scavenger.

References Cited R O R UNITED STATES PATENTS 3 o 011 3,458,628 7/1969 Kaiser et al. 260-2105 em 3,462,528 8/1969 Voigtlander etal. 260210.5 3,472,836 10/1969 Vogelsang et al. 260-210.5 3,546,208 12/1970 Steidle 260-2105 3,585,187 6/1971 Heider et al. 2602l0.5

wherein R is methyl or formyl; R and R are hydrogen, LEWIS GOTTS Primary Examiner and R is lower alkyl; which process comprises contacting a A -bufatrienolide rha'mnoside of the formula R BROWN, Asslstant EXamlIlel' US. Cl. X.R. A 424-482 D H3O CH3 40 

